Activation of the HIV-1 protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activity requires the formation of protease homodimers. Mutations that block dimerization interfere with the protease function; viral variants encoding non-functional enzymes are aberrantly assembled and non-infectious. In vitro studies of compounds that inhibit the enzyme from dimerizing produce similar results. Despite the invariant nature of protease dimerization across all retroviral systems, important biological and structural questions remain unanswered. First, although structural studies have identified the residues involved in the dimer interface, the role of specific amino acids in promoting and maintaining dimer formation has not been examined. Second, a wealth of structural and biological information suggests that there is a close association between ordered, protease-mediated precursor processing, particle assembly and infectivity. However, the precise effect of protease dimerization and subsequent enzyme activation on particle assembly and infectivity is unclear. Finally, although clinically available substrate-based inhibitors of the HIV protease have made a dramatic impact on disease progression, resistant variants frequently arise in patients treated with these active site-directed compounds. Novel approaches to inhibitor design are urgently needed to develop additional effective therapeutic agents. In the studies described below, we will define the interactions critical for maintenance of the HIV protease dimer and the role that the final structure plays in viral replication. Further, we will explore novel strategies for inhibition of the viral protease through disrupting dimer formation. Overall, our studies will provide important insights into protease structure, enzyme function and retroviral biology. Specifically, we will: I. Define the role of individual amino acids in dimer formation. II. Characterize the effect of substitutions in the F-IIV protease dimer interface on viral replication. IlI. Characterize HIV protease activation within GagPol as a potential target for inhibition.